Generation of long insert pairs using a Cre-LoxP Inverse PCR approach

Large insert mate pair reads have a major impact on the overall success of de novo assembly and the discovery of inherited and acquired structural variants. The positional information of mate pair reads generally improves genome assembly by resolving repeat elements and/or ordering contigs. Currently available methods for building such libraries have one or more of limitations, such as relatively small insert size; unable to distinguish the junction of two ends; and/or low throughput. We developed a new approach, Cre-LoxP Inverse PCR Paired-End (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) the use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends and to improve the self-ligation efficiency. We have successfully created CLIP-PE libraries up to 22 kb that are rich in informative read pairs and low in small fragment background. These libraries have demonstrated the ability to improve genome assemblies. The CLIP-PE methodology can be implemented with existing and future next-generation sequencing platforms.     

PI3K signalling: the path to discovery and understanding

Over the past two decades, our understanding of phospoinositide 3-kinases (PI3Ks) has progressed from the identification of an enzymatic activity associated with growth factors, GPCRs and certain oncogene products to a disease target in cancer and inflammation, with PI3K inhibitors currently in clinical trials. Elucidation of PI3K-dependent networks led to the discovery of the phosphoinositide-binding PH, PX and FYVE domains as conduits of intracellular lipid signalling, the determination of the molecular function of the tumour suppressor PTEN and the identification of AKT and mTOR protein kinases as key regulators of cell growth. Here we look back at the main discoveries that shaped the PI3K field.     

Intramuscular temperatures during exercise in the heat following pre-cooling and pre-heating

Pre-cooling improves heat tolerance and time to exhaustion in the heat. We tested the possibility that reduced tissue temperatures may explain this phenomenon, using three whole-body treatments: pre-cooling, thermoneutral (control) and pre-heating. Pre-cooling reduced muscle temperature (T_m) by 6.3 °C while pre-heating increased T_m 3.4 °C, relative to control. Despite this offset, T_m climbed towards a common asymptote, with pre-cooling offering no thermal protection beyond 40 min. Following pre-cooling, exercising oesophageal temperature (T_es) initially increased at 0.09 °C min⁻¹, being significantly faster than control (0.05 °C min⁻¹) and pre-heated conditions (0.03 °C min⁻¹). Pre-cooling lowered the sweat threshold and also resulted in a reduced cardiac frequency across the exercise-heat exposure. Our observations do not support the hypothesis that pre-cooling reduces T_m at the end of an exercise-heat exposure, thereby delaying the development of fatigue.